Holotype HLA Early Access Program Results: An “Uncontrolled” Study

EFI-2016-EAP-Poster-768x157

Authors: Efi Melista, Krisztina Rigó, Szilveszter Juhos, György Horváth, Peter Meintjes, Tim Hague

Abstract:

NGS-based (Next Generation Sequencing) HLA typing can offer accurate and unambiguous genotyping results with minimal manual intervention, eliminating the requirement for reflexive testing. In a clinical setting an easy-to-follow protocol with minimal hands-on time is also essential. Holotype HLA is an optimised, pre-configured assay and software combination product that provides comprehensive gene characterisation of multiple HLA loci for sequencing on the Illumina MiSeq. The assay provides a streamlined, high-throughput, fully automatable protocol that is designed specifically for clinical lab implementation.

The software, HLA Twin, uses two independent algorithms and 15 informative Quality Control metrics for confident allelic determination. The Early Access Program was launched in October 2014 and run for 9 months.

The goals were to introduce Holotype HLA and NGS to the HLA community, and to show its reproducibility, high sensi- tivity and high accuracy. The participants were 24 different labs from 13 countries worldwide. Most of the labs had never seen the Holotype HLA protocol before and some had no prior experience in sequencing. A total of 2530 samples were genotyped for a minimum of 5 loci (HLA-A, HLA-B, HLA-C, HLA-DQB1 and HLA-DRB1). In this process 26282 alleles were identified of which 8788 alleles had known typings generated with a previous technology (SBT or SSO/SSP). Some of the metrics we focused on were the concordance rate, ambiguity rate (as well as the causes of ambiguity) and accuracy.

Overall, 96.7% concordance with reference typings was identified. Of the discordant samples, 1.7% were due to an incorrect reference typing, 0.7% were due to a novel allele and the remaining were due to low quality data indicated by QC failures. No ambiguities were identified for results up to the 3rd field, except in 6 instances for HLA-DRB1. The cause of these ambiguities was due to variations located in off-target amplification regions. Finally, the accuracy of the results was more than 99.9% for HLA-A, HLA-B, HLA-C and HLA-DRB1 and 99.76% for HLA-DQB1. The cause of the lower accuracy rate for HLA-DQB1 has been identified and successfully improved since the end of the EAP.

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